Project: The evolutionary genetics and genomics of Wolbachia effects on host physiology Project Summary: Insects are the most abundant group of organisms on the planet, and the cells of about half of these species are infected with maternally transmitted Wolbachia bacteria. Wolbachia became recognized for manipulating host reproduction; for example, many Wolbachia strains cause cytoplasmic incompatibility (CI), which generates increased embryo mortality when Wolbachia-infected males mate with uninfected females. Several Wolbachia strains that infect Drosophila flies also block viruses, and when introgressed into Aedes aegypti, the wMel strain that naturally infects D. melanogaster serves as a biocontrol of vector-borne disease (particularly dengue and now Zika). Despite the initial success of Wolbachia biocontrol, large gaps in knowledge exist regarding the conditions that favor Wolbachia infections. For example, in no Wolbachia-infected host system is it understood how Wolbachia infections positively affect host fitness to spread from low frequencies. This is surprising considering that Wolbachia have spread to become the most prevalent symbiont on the planet. The investigator's long-term goal is to understand the genetic and abiotic contexts that favor the spread and persistence of Wolbachia infections. The central hypothesis is that interactions among host nuclear, host mitochondrial, and Wolbachia genomes (and the environment) determine the fitness effects of Wolbachia on hosts. The rationale is that leveraging naturally occurring genetic variation, in combination with integrative approaches and new technology, now enables these gaps in knowledge to be filled. Guided by preliminary data and well-established theory, the proposed research will test the central hypothesis by determining contributions of genomic and environmental interactions to: 1) variation in CI, cell physiology, and fitness; and 2) variation in the abundance and distribution of Wolbachia cells within host tissues, which underlies efficient maternal Wolbachia transmission to host offspring. Naturally sampled genetic variation and genotypes constructed in the lab will be used alongside next-generation sequencing to answer questions. For example, preliminary analyses have identified both host backgrounds and Wolbachia genomic variants that influence the focal traits. The proposed projects will use Wolbachia microinjections to reciprocally introgress candidate Wolbachia and host backgrounds, some of which have precise combinations of nuclear and mitochondrial genomes. This design enables an explicit test for contributions of different genomes to variation in phenotypes that affect fitness. Regarding point two above, recent data indicate that wMel transmission is highly susceptible to temperature in both fly and mosquito backgrounds. The proposed projects will use analytical chemistry and confocal microscopy to understand the cell basis of this susceptibility. The proposed research is significant because it will identify the genetic and abiotic contexts that facilitate Wolbachia spread. The proposed research is innovative because it bridges gaps between studies of genomes, cells, and natural genetic variation; these gaps currently inhibit understanding Wolbachia persistence in nature.